This invention relates to a method for measuring bilirubin contained in a living body fluid such as plasma, serum, urine or the like.
Bilirubin is a compound derived from hemoglobin liberated by lysis of aged erythrocyte. Blood of a living body contains direct bilirubin (mainly glucuronide conjugate form) which reacts directly with a diazo compound to develop a color, and indirect bilirubin (free form) which reacts with a diazo compound only in the presence of an accelerator such as an alcohol.
Diagnoses of various liver diseases and diacrisis of jaundice can be made by measuring total bilirubin concentration and the concentration of each of the above two forms of bilirubin in blood, and hence the measurement of bilirubin is an important examination among clinical examinations.
At present, a leading method for measuring bilirubin is the so-called diazo method which comprises measuring the purplish red color of azobilirubin formed by the reaction of bilirubin with a diazo compound. The diagnoses of various liver diseases and the diacrisis of jaundice are made on the basis of the concentrations of bilirubins measured mainly by this method. This method, however, is disadvantageous, for example, in that reagent solutions for the method can be used for only about 5 days after their preparation, and that measured values are affected by L-ascorbic acid and hemoglobin which are present in a sample.
As other methods for measuring bilirubin, there can be exemplified BOD method utilizing bilirubin oxidase (BOD) as an oxidizing agent, high-performance liquid chromatography method utilizing high-performance liquid chromatography, film method comprising measuring .alpha., .beta., .GAMMA. and .delta. forms of bilirubin by use of a film coated with a mordant, and chemical oxidation method using ferricyanide ions or copper ions as an oxidizing agent (e.g. a method for measuring total or direct bilirubin by use of potassium ferricyanide, a method for measuring direct bilirubin by use of copper ions and thiourea, etc.). All of these methods involve some problem and cannot be said to be preferable to the diazo method.
In detail, the BOD method involves no particular problem when used for measuring total bilirubins, but is disadvantageous in that when it is used for measuring direct bilirubin, the measurement should be carried out at a pH outside the optimum pH range for BOD, so that a large amount of the enzyme is unavoidably needed, resulting in a high cost of the measurement. The high-performance liquid chromatography method and the film method are rated high in the precision of measurement but are disadvantageous, for example, in that they require an apparatus for exclusive use and cannot be utilized for measuring many items at the same time for a large number of samples. In the case of the chemical oxidation method, total bilirubins and direct bilirubin cannot be measured at the same pH, so that the measurement is influenced by a change of the absorption curve of bilirubin which accompanies a pH change. Therefore when direct bilirubin is measured utilizing an absorbance value measured by use of standard solutions at the time of measuring total bilirubins, the reliability of measured values is lowered. Furthermore, the BOD method and the chemical oxidation method are disadvantageous also in that a measured value of direct bilirubin obtained by these methods is in poor correlation with that obtained by the diazo method, and hence is difficult to utilize for diagnoses of various liver diseases and diacrisis of jaundice which have been established on the basis of measured values obtained by the diazo method.
Under such circumstances, there is desired a method for measuring bilirubin which can be applied to an automatic analyzer, is in good correlation with the diazo method, and is excellent in the stability of reagent solutions.